PubMed Health⌕ Search

Biomedical subjects

C Dargemont

Publications and source records attributed to C Dargemont.

34 records · Page 2Linked to original sources
PubMed1999

Direct association and nuclear import of the hepatitis B virus X protein with the NF-kappaB inhibitor IkappaBalpha.

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.

Animals↗
PubMed1997

Evidence for a role of CRM1 in signal-mediated nuclear protein export.

Chromosome maintenance region 1 (CRM1), a protein that shares sequence similarities with the karyopherin beta family of proteins involved in nuclear import pathway, was shown to form a complex with the leucine-rich nuclear export signal (NES). This interaction was inhibited by leptomycin B, a drug that prevents the function of the CRM1 protein in yeast. To analyze the role of the CRM1-NES interaction in nuclear export, a transport assay based on semipermeabilized cells was developed. In this system, which reconstituted NES-, cytosol-, and energy-dependent nuclear export, leptomycin B specifically blocked export of NES-containing proteins. Thus, the CRM1 protein could act as a NES receptor involved in nuclear protein export.

Adenosine Triphosphate↗
PubMed1997

Nuclear Rel-A and c-Rel protein complexes are differentially distributed within human thymocytes.

Nuclear factor-kappa B (NF-kappa B)/Rel proteins are inducible transcriptional regulators of numerous cellular genes. They are particularly abundant in lymphoid tissues and are thought to be critical for the transcription of genes involved in immune and inflammatory responses. We have reported previously that a nuclear NF-kappa B activity was present in freshly extracted human thymocytes in the absence of in vitro treatment of these cells. In the present report, we identified NF-kappa B proteins extracted from human thymocyte nuclei as being p50/p65 and p50/c-Rel complexes. Immunochemical and immunofluorescent staining of thymus sections using specific Abs allowed visualization of nuclear NF-kappa B proteins in both thymocytes and nonthymocyte cells. This detection suggested a preferential activation of p50/c-Rel in medullary thymocytes, whereas p50/p65 was present in both cortical and medullary regions of human thymus lobules. However, the intensity of p65 labeling was much higher in several thymocytes from the medulla. p65, p50, and c-Rel activities were found in both CD4- and CD8-positive thymocytes. These observations suggest that p65 and c-Rel complexes play distinct roles in gene expression and that both forms of NF-kappa B play critical roles during late stages of the intrathymic maturation of T cells.

Cell Nucleus↗
PubMed1997

Nuclear export signal of IkappaBalpha interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I.

De novo synthesized IkappaBalpha accumulates transiently in the nucleus where it inhibits NF-kappaB-dependent transcription and reduces nuclear NF-kappaB content. A sequence present in the C-terminal domain of IkappaBalpha and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IkappaBalpha the ability to export IkappaBalpha/NF-kappaB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IkappaBalpha could interfere with Rev-dependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IkappaBalpha in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IkappaBalpha to inhibit NF-kappaB-DNA-binding activity and required a functional IkappaBalpha NES motif. Indeed, mutation of the NES abrogated the capacity of IkappaBalpha to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-kappaB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IkappaBalpha NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IkappaBalpha, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-kappaB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-kappaB is likely the consequence of interference in the IkappaBalpha-mediated export of NF-kappaB. These findings indicate that IkappaBalpha and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IkappaBalpha, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.

Animals↗
PubMed1997

Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm.

I kappa B alpha tightly regulates the transcriptional activity of NF-kappa B by retaining it in the cytoplasm in an inactive form. In the present work, we report that I kappa B alpha, when expressed in the nuclear compartment, not only abrogates NF-kappa B/DNA interactions and NF-kappa B-dependent transcription, but also transports NF-kappa B back to the cytoplasm. This function of I kappa B alpha is insured by a nuclear export sequence located in the C-terminal domain of I kappa B alpha and homologous to the previously described export signal found in HIV-1 Rev protein as well as in PKI (the inhibitor of the catalytic subunit of protein kinase A). Thus, inhibition of NF-kappa B/DNA binding and the consecutive efficient nuclear export of the transcription factor of I kappa B alpha could represent an important mechanism for the control of the expression of NF-kappa B-dependent genes.

Amino Acid Sequence↗
PubMed1996

p95vav associates with the nuclear protein Ku-70.

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.

Base Sequence↗
PubMed1995

Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus.

Primary transcripts in eukaryotic cells undergo several processing steps within the nucleus, and resulting mature RNA molecules are selectively exported to the cytoplasm. Nucleo-cytoplasmic mRNA transport is an active process that likely involves RNA-protein interactions. To identify specific RNA-binding proteins, we designed a novel approach, which allows the analysis of interactions between mRNAs and proteins along the transport pathway. The method consists of inducing in vivo a covalent binding between nuclear proteins and microinjected mRNAs. Using such a procedure, we were able to detect a direct interaction between nucleoporin p62 with mRNA during export. The formation of the mRNA-p62 complex was inhibited by wheat-germ agglutinin, an inhibitor of mRNA export. Antibodies directed against p62 caused a substantial reduction in the rate of mRNA export from the nucleus.

Animals↗
PubMed1993

Nuclear export of proteins: the role of nuclear retention.

Proteins that shuttle between nucleus and cytoplasm are implicated in transport and signal transduction processes. Using assays based on interspecies heterokaryons and microinjection of Xenopus oocytes, we examined what structural features determine nuclear export of shuttling proteins. Three classes of proteins were studied: first, wild-type and mutant forms of nucleolin, one of the first shuttling proteins identified; second, artificial nuclear reporter proteins derived from cytoplasmic pyruvate kinase; and third, wild-type and mutant lamins differing in their abilities to be incorporated into the lamina. Our results show that a protein does not require positively acting export signals to be transported from nucleus to cytoplasm; instead, its shuttling ability is limited primarily by intranuclear interactions. We conclude that nucleocytoplasmic shuttling is a general phenomenon not restricted to proteins involved in nucleocytoplasmic transport.

Amino Acid Sequence↗
PubMed1993

The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.

Amino Acid Sequence↗
PubMed1993

Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow.

The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre-established mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whitlock/Witte-type culture) or in the presence of 10(-7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cells accounting for 50% to 90% of the organ-cultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3-/low and TCR alpha beta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus.

Animals↗
PubMed1992

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

Adenosine Triphosphate↗
PubMed1991

Overproduction and secretion of beta 2-microglobulin by a rat thymic epithelial cell line that expresses MHC class I heavy chain.

Major histocompatibility complex (MHC) class I antigens are constituted of dimers consisting of a peripheral light chain, beta 2-microglobulin (beta 2m) and a transmembrane heavy chain whose cell surface expression depends on its assembly with beta 2m. In contrast, soluble beta 2m can be secreted in the absence of heavy chain expression. The presence of beta 2m in medium conditioned by a rat thymic epithelial cell line, IT45-R1 (IT45) prompted us to investigate whether beta 2m could be secreted by cells that express MHC class I antigens. IT45 cells produce three to five times more beta 2m in the culture supernatant than another rat thymic epithelial cell line, IT26-R21 (IT26). The IT45 cell line exported beta 2m through a constitutive pathway of secretion, as indicated by the kinetics of production and localization of intracellular beta 2m. Although cells from the IT45 cell line expressed a much higher amount of beta 2m as compared to IT26 and NBT II cells (a rat bladder epithelial cell line), all three of these cell lines expressed the same amount of membrane and intracellular MHC class I heavy chain. These data are thus consistent with a constitutive secretion of beta 2m dependent upon an overexpression of MHC class I light chain as compared to the heavy chain. The amount of beta 2m mRNA and the ratio of beta 2m versus MHC class I heavy chain transcripts were higher in IT45 than in IT26 cells, indicating that overexpression of beta 2m in IT45 cells could be due to an enhanced level of beta 2m mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Rat bone marrow cells undergo thymopoiesis in mouse fetal thymic organ culture.

We have developed an in vitro differentiation assay allowing the study of thymopoiesis from rat bone marrow cells. In this assay, Wistar rat bone marrow cells repopulated fetal Swiss mouse thymic lobes depleted in endogeneous lymphoid cells by deoxyguanosine treatment. Due to the xenogeneic situation, repopulating rat cells from any hemopoietic lineage could be easily recognized by anti-rat monoclonal antibodies such as anti-Thy-1.1 that did not react with Swiss mouse thymocytes. After 15 days in vitro, 80% of the developing rat cells were Thy-1.1+ lymphoid cells and about 70% of the Thy-1.1+ cells expressed CD5, CD2 and leukosialin. The percentages of cells expressing pre-B cell, B cell and myeloid determinants were less than 20%. The developing thymocytes comprised CD4-CD8- T cell receptor (TcR) alpha/beta-, CD4-CD8+TcR alpha/beta low and CD4+CD8+TcR alpha/beta low cells, indicating that the early stages of rat thymopoiesis occurred within mouse thymic lobes. Limiting dilution assays showed that 50% of positive assays were obtained with 3000 nucleated bone marrow cells, which is in good agreement with recent estimates derived from in vivo reconstitution after intrathymical transfer. Moreover the limiting dilution assays proved to be sensitive enough to evidence a tenfold enrichment of pre-T cell activity in the low-density fraction of rat bone marrow. This xenogeneic system might greatly facilitate studies on prethymic and intrathymic stages of rat T cell development and permit new in vitro approaches of the colonizing bone marrow T cell precursor properties.

Animals↗
PubMed1989

Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin.

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.

Animals↗
PubMed1988

Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles.

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

Adenosine Triphosphate↗